Browsing by Author "Costa, Pedro M."
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- An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responsesPublication . Costa, Pedro M.; Pinto, Miguel; Vicente, Ana; Gonçalves, Cátia; Rodrigo, Ana; Louro, Henriqueta; Costa, Maria Helena; Caeiro, Sandra; Silva, Maria JoãoThe application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors, e.g., when dealing with estuarine sediments, that hampers the drawing of cause-effect relationships. Although the in vitro Comet assay may circumvent confounding factors, its application in environmental risk assessment (ERA) still needs validation. As such, the present work aims at integrating genotoxicity and oxidative DNA damage induced by sediment-bound toxicants in HepG2 cells with oxidative stress-related effects observed in three species collected from an impacted estuary. Distinct patterns were observed in cells exposed to crude mixtures of sediment contaminants from the urban/industrial area comparatively to the ones from the rural/riverine area of the estuary, with respect to oxidative DNA damage and oxidative DNA damage. The extracts obtained with the most polar solvent and the crude extracts caused the most significant oxidative DNA damage in HepG2 cells, as measured by the formamidopyrimidine-DNA glycosylase (FPG)-modified Comet assay. This observation suggests that metals and unknown toxicants more hydrophilic than polycyclic aromatic hydrocarbons may be important causative agents, especially in samples from the rural part of the estuary, where oxidative DNA damage was the most significant. Clams, sole, and cuttlefish responded differentially to environmental agents triggering oxidative stress, albeit yielding results accordant with the oxidative DNA damage observed in HepG2 cells. Overall, the integration of in vivo biomarker responses and Comet assay data in HepG2 cells yielded a comparable pattern, indicating that the in vitro FPG-modified Comet assay may be an effective and complementary line-of-evidence in ERA even in particularly challenging, natural, scenarios such as estuarine environments.
- Assessment of sediment contamination in an impacted estuary: differential effects and adaptations of sentinel organisms and implications for biomonitoringPublication . Costa, Pedro M.; Gonçalves, C.; Martins, M.; Rodrigo, Ana; Carreira, S.; Costa, Maria Helena; Caeiro, SandraEstuarine pollution is reflected in the concentration of toxicants in sediments, depending on their geochemical properties, since sediments trap substances from the water column, either dissolved or bound to suspended matter. However, determining risk of sediment contaminants to biota has many constraints. For such reason, integrative approaches are keystone. Taking the Sado estuary (SW Portugal) as a case study, contrasted to a reference estuary (the Mira) within the same geographical location, the present study aimed at integrating sediment contamination with the effects and responses to pollutants in distinct benthic organisms with commercial and ecological value.
- Assessment of the genotoxic potential of contaminated estuarine sediments in fish peripheral blood: laboratory versus in situ studiesPublication . Costa, Pedro M.; Neuparth, Teresa S.; Caeiro, Sandra; Lobo, Jorge; Martins, Marta; Ferreira, Ana M.; Caetano, Miguel; Vale, Carlos; DelValls, T. Ángel; Costa, Maria HelenaJuvenile Senegalese soles (Solea senegalensis) were exposed to estuarine sediments through 28-day laboratory and in situ (field) bioassays. The sediments, collected from three distinct sites (a reference plus two contaminated) of the Sado Estuary (W Portugal) were characterized for total organic matter, redox potential, fine fraction and for the levels of metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorines, namely polychlorinated biphenyls (PCBs) and dichloro diphenyl tricholoethane plus its main metabolites (DDTs). Genotoxicity was determined in whole peripheral blood by the single-cell gel electrophoresis (SCGE or ‘‘comet’’) assay and by scoring erythrocytic nuclear abnormalities (ENA). Analysis was complemented with the determination of lipid peroxidation in blood plasma by the thiobarbituric acid reactive substances (TBARS) protocol and cell type sorting. The results showed that exposure to contaminated sediments induced DNA fragmentation and clastogenesis. Still, laboratory exposure to the most contaminated sediment revealed a possible antagonistic effect between metallic and organic contaminants that might have been enhanced by increased bioavailability. The laboratory assay caused a more pronounced increase in ENA whereas a very significant increase in DNA fragmentation was observed in field-tested fish exposed to the reference sediment, which is likely linked to increased lipid peroxidation that probably occurred due to impaired access to food. Influence of natural pathogens was ruled out by unaltered leukocyte counts. The statistical integration of data correlated lipid peroxidation with biological variables such as fish length and weight, whereas the genotoxicity biomarkers were more correlated to sediment contamination. It was demonstrated that laboratory and field bioassays for the risk assessment of sediment contamination may yield different genotoxicity profiles although both provided results that are in overall accordance with sediment contamination levels. While field assays may provide more ecologically relevant data, the multiple environmental variables may produce sufficient background noise to mask the true effects of contamination.
- Avaliação do potencial citotóxico e genotóxico de contaminantes de sedimentos do Estuário do Sado numa linha celular humanaPublication . Pinto, Miguel; Costa, Pedro M.; Louro, Henriqueta; Costa, Maria Helena; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoO presente estudo integra-se num projeto mais amplo que visa avaliar o risco ambiental – que inclui os riscos ecológicos e para a saúde humana - associado a este ambiente estuarino contaminado. Em particular, este estudo teve como objetivo caracterizar o potencial citotóxico e genotóxico de sedimentos colhidos em vários locais de pesca do Estuário do Sado numa linha celular humana, tendo em vista uma avaliação de eventuais efeitos nefastos para a saúde humana.
- Biochemical endpoints on juvenile Solea senegalensis exposedPublication . Costa, Pedro M.; Caeiro, Sandra; Diniz, Mário S.; Lobo, Jorge; Martins, Marta; Ferreira, Ana M.; Caetano, Miguel; Vale, Carlos; DelValls, T. Ángel; Costa, Maria HelenaJuvenile Solea senegalensis were exposed to fresh sediments from three stations of the Sado estuary (Portugal) in 28-day laboratory assays. Sediments revealed distinct levels of total organic matter, fine fraction, redox potential, trace elements (arsenic, cadmium, chromium, copper, nickel, lead and zinc) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide: dichloro diphenyl trichloroethane). Organisms were surveyed for contaminant bioaccumulation and induction of two hepatic biochemical biomarkers: metallothionein (MT) and cytochrome P450 (CYP1A), as potential indicators of exposure to metallic and organic contaminants, respectively. Using an integrative approach it was established that, although bioaccumulation is in general accordance with sediment contamination, lethality and biomarker responses are not linearly dependent of the cumulative concentrations of sediment contaminants but rather of their bioavailability and synergistic effects in organisms. It is concluded that metals and organic contaminants modulate both MT and CYP1A induction and it is suggested that reactive oxygen species may be the link between responses and effects of toxicity.
- Can the integration of multiple biomarkers and sediment geochemistry aid solving the complexity of sediment risk assessment?: a case study with a benthic fishPublication . Costa, Pedro M.; Caeiro, Sandra; Vale, Carlos; Delvalls, T. Ángel; Costa, Maria HelenaSurveying toxicity of complex geochemical media as aquatic sediments often yields results that are either difficult to interpret or even contradictory to acknowledged theory. Multi-level biomarkers were investigated in a benthic fish exposed to estuarine sediments through laboratory and in situ bioassays, to evaluate their employment either in ecological risk assessment or in more mechanistic approaches to assess sediment-bound toxicity. Biomarkers reflecting lesions (such as genotoxicity or histopathology), regardless of their low or absent specificity to contaminants, are efficient in segregating exposure to contaminated from uncontaminated sediments even when classical biomarkers like CYP1A and metallothionein induction are inconclusive. Conversely, proteomics and gene transcription analyses provided information on the mechanics of toxicity and aided explaining response variation as a function of metabolic imbalance and impairment of defences against insult. In situ bioassays, although less expedite and more affected by confounding factors, produced data better correlated to overall sediment contamination.
- A description of chloride cell and kidney tubule alterations in the flatfish Solea senegalensis exposed to moderately contaminated sediments from the Sado estuary (Portugal)Publication . Costa, Pedro M.; Caeiro, Sandra; Diniz, Mário S.; Lobo, Jorge; Martins, Marta; Ferreira, Ana M.; Caetano, Miguel; Vale, Carlos; DelValls, T. Ángel; Costa, Maria HelenaThe effects of sediment-bound contaminants on kidney and gill chloride cells were surveyed in juvenile Solea senegalensis exposed to fresh sediments collected from three distinct sites of the Sado Estuary (Portugal) in a 28-day laboratorial assay. Sediments were analyzed for metallic contaminants, polycyclic aromatic hydrocarbons and organochlorines as well as for total organic matter, redox potential and fine fraction. The potential for causing adverse biological effects of each surveyed sediment was assessed by comparison of contaminant levels to available guidelines for coastal sediments, namely the Threshold Effects Level (TEL) and the Probable Effects Level (PEL). The Sediment Quality Guideline Quotient indices (SQGQ) were calculated to compare the overall contamination levels of the three stations. A qualitative approach was employed to analyze the histo/cytopathological traits in gill chloride cells and body kidney of fish exposed to each tested sediment for 0, 14 and 28 days. The results showed that sediment contamination can be considered low to moderate and that the least contaminated sediment (from a reference site, with the lowest SQGQ) caused lesser changes in the surveyed organs. However, the most contaminated sediment (by both metallic and organic xenobiotics, with highest SQGQ) was neither responsible for the highest mortality nor for the most pronounced lesions. Exposure to the sediment presenting an intermediate SQGQ, essentially contaminated by organic compounds, caused the highest mortality (48%) and the most severe damage to kidneys, up to full renal necrosis. Chloride cell alterations were similar in fish exposed to the two most contaminated sediments and consisted of a pronounced cellular hypertrophy, likely involving fluid retention and loss of mitochondria. It can be concluded that sediment contamination considered to be low or moderate may be responsible for severe injury to cells and parenchyma involved in the maintenance of osmotic balance, contributing for the high mortality levels observed. The results suggest that sediment-bound organic contaminants such as PAHs (polycyclic aromatic hydrocarbons) and PCBs (polychlorinated biphenyls) may be very toxic to the analyzed organs, especially the kidney, even when present in lowrisk concentrations.
- Determining oxidative and non-oxidative genotoxic effects driven by estuarine sediment contaminants on a human hepatoma cell linePublication . Costa, Pedro M.; Louro, Henriqueta; Costa, Maria Helena; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoEstuarine sediments may be reservoirs of hydrophilic and hydrophobic pollutants, many of which are acknowl- edged genotoxicants, pro-mutagens and even potential carcinogens for humans. Still, studies aiming at narrowing the gap between ecological and human health risk of sediment-bound contaminant mixtures are scarce. Taking an impacted estuary as a case study (the Sado, SW Portugal), HepG2 (human hepatoma) cells were exposed in vitro for 48 h to extracts of sediments collected from two areas (urban/industrial and Triverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, among which are found priority mutagens such as benzo[a]pyrene. Comparatively to a control test, extracts of sediments from both impacted areas produced deleterious effects in a dose–response manner. However, sediment extracts from the industrial area caused lower replication index plus higher cytotoxicity and genotoxicity (concerning total DNA strand breakage and clastogenesis), with emphasis on micronucleus induction. On the other hand, extracts from the rural area induced the highest oxidative damage to DNA, as revealed by the FPG (formamidopyrimidine-DNA glycosylase) enzyme in the Comet assay. Although the estuary, on its whole, has been classified as moderately contaminated, the results suggest that the sediments from the industrial area are significantly genotoxic and, furthermore, elicit permanent chromosome damage, thus potentially being more mutagenic than those from the rural area. The results are consistent with contamination by pro-mutagens like polycyclic aromatic hydrocarbons (PAHs), potentiated by metals. The sediments from the agriculture-influenced area likely owe their genotoxic effects to metals and other toxicants, probably pesticides and fertilizers, and able to induce reactive oxygen species without the formation of DNA strand breakage. The findings suggest that the mixtures of contaminants present in the assayed sediments are genotoxic to HepG2 cells, ultimately providing a useful approach to hazard identification and an effective line-of-evidence in the environmental monitoring of anthropogenically-impacted coastal ecosystems.
- Development of histopathological indices in a commercial marine bivalvePublication . Costa, Pedro M.; Carreira, S.; Costa, Maria Helena; Caeiro, SandraBivalve histopathology is an acknowledged tool in environmental toxicology studies, however geographically restricted, limited to a few species and still lacking the degree of detail needed to develop effective (semi)quantitative approaches. A first-time detailed histopathological screening was performed on grooved carpet shell clams collected from commercial shellfish beds in distinct coastal ecosystems of the Southern Portuguese coast: two parted sites within an impacted estuary (S1 and S2), an inlet channel of a fish farm at a considered pristine estuary (site M) and a site allocated in a clean coastal lagoon (A). A total of thirty histopathological lesions and alterations were analysed in the gills and digestive glands following a weighted condition indices approach, including inflammation-related responses, necrosis, neoplastic diseases and parasites. Digestive glands were consistently more damaged than gills, except for animals collected from site M, where the most severe lesions were found in both organs, immediately followed by S2. Clams from sites S1 and A were overall the least damaged. Neoplastic diseases were infrequent in all cases. Inflammation-related traits were some of the most common alterations progressing in animals enduring severe lesions such as digestive tubule (diverticula) and intertubular tissue necrosis. Some alterations, such as lipofuscin aggregates within digestive tubule cells, did not relate to histological lesions. Granulocytomas only occurred in heavily infected tissues. Animals from M and A presented the highest infections in the digestive gland, especially by protozoa. Gill infections were more similar between sites. Still, the level of infection does not account for all histopathological lesions in either organ. Overall, the results are in accordance with environmental parameters, such as distance to pollution sources, sediment type and hydrodynamics, and show that the combination of multiple histopathological features in these clams provides good sensitivity for inter-site distinction even when low or moderate anthropogenic impacts are at stake.
- Ecological risk assessment of impacted estuarine areas: integratingPublication . Gonçalves, Cátia; Martins, M.; Costa, Maria Helena; Caeiro, Sandra; Costa, Pedro M.The analysis of multiple biomarker responses is nowadays recognized as a valuable tool to circumvent potential confounding factors affecting biomonitoring studies and allows a better understanding of the mechanisms underlying exposure to natural mixtures of toxicants. For the purpose of an environmental risk assessment (ERA) in an impacted estuary in SW Europe (the Sado, Portugal), juvenile Solea senegalensis from commercial fishing areas were surveyed for histopathological liver alterations and biochemical biomarkers. Although the findings revealed moderate differences in the patterns of histopathological traits between urban/industrial- and agricultural-influenced areas within the same estuary, no significant distinction was found between the cumulative alterations in animals from the two sites. The overall level of histopathological injury was low and severe traits like neoplasms or preneoplastic foci were absent. While metallothionein induction and lipid peroxidation could relate to histopathological condition indices, the activity of anti-oxidant enzymes appeared to be impaired in animals collected off the estuary's heavy-industry belt (the most contaminated site), which may partially explain some degree of hepatic integrity loss. Overall, the results are consistent with low-moderate contamination of the estuary and indicate that oxidative stress is the most important factor accounting for differences between sites. The study highlights the need of integrating multiple biomarkers when multiple environmental stressors are involved and the advantages of surveying toxicity effects in fieldcollected, foraging, organisms.